Antimicrobial Alkaloid Research Articles

The pygidial defense gland system of the Steninae (Coleoptera, Staphylinidae): Morphology, ultrastructure and evolution

The pygidial defense glands of the Steninae consist of two big (r1) and two smaller (r2) secretion filled sac-like reservoirs with associated secretory tissues and basal eversible membrane structures. The secretion is made up of deterrent and antimicrobial alkaloids stored in r1 as well as terpenes in r2. The gland cells filling r1 form a band shaped secretory tissue (g1) in an invagination of the reservoir membrane. The content of r2 is secreted by a tissue (g2) surrounding the efferent duct of r1 opposite to r2. In both gland tissues the secretion is produced in type IIIt gland cells and accumulates in an extracellular cavity surrounded by numerous microvilli of the gland cell membrane. After exocytosis the secretion enters an epicuticular duct and is transported to the corresponding reservoir via a conducting canal enclosed in at least one canal cell. While the structure of g1 is very similar in all species of the Steninae, g2 is often reduced. This reduction of the system r2/g2 is accompanied by a decreasing amount of terpenes in the total secretion and could be of interest for phylogenetic studies in the subfamily of the Steninae.

Identification and antimicrobial activity of two alkaloids from traditional Chinese medicinal plant Tinospora capillipes

Two antimicrobial alkaloids, palmatine and jatrorrhizine, were isolated from tubers of traditional Chinese medicinal plant Tinospora capillipes using activity-guided isolation method and chromatography. Their antimicrobial activity was determined in vitro. The results showed that palmatine and jatrorrhizine had inhibitory activity against plant pathogens Colletotrichum gloeosporioides, Fusarium oxysporum f. sp. niveum, Mycosphaerella sentina, Pestalotia mangiferae, Cercospora kaki, Gymnosporangium haraeanum, Rhizoctonia solani and Colletotrichum graminicola, with the EC50 values of 0.0348–0.8356gL−1 and 0.0240–0.8649gL−1, respectively. Palmatine and jatrorrhizine also exhibited inhibition against animal pathogens Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Staphyloccocus aureus, Staphylococcus epidermidi, Micrococcus lysodeikticus, Proteus vulgaris, Salmonella typhi and Escherichia coli, with the MIC values of 0.1–0.8gL−1 and 0.1–0.6gL−1, respectively. These results suggested that palmatine and jatrorrhizine showed relatively broad spectrum antimicrobial activity against plant and animal pathogens.

An antimicrobial alkaloid and other metabolites produced by Penicillium sp. An endophytic fungus isolated from Mauritia flexuosa L. f.

The alkaloid glandicoline B (1) and six other compounds: ergosterol (2), brassicasterol (3), ergosterol peroxide (4), cerevisterol (5), mannitol (6) and 1-O-α-D-glucopyranoside (7) were isolated from Penicillium sp. strain PBR.2.2.2, a fungus from Mauritia flexuosa roots. The structures of the isolated metabolites were established by spectral analysis. MeOH extract of the fungal mycelium at 500 µg mL-1 exhibited antimicrobial activity against Staphylococcus aureus and the compound 1 at 100 µg mL-1 was active against S. aureus, Micrococcus luteus and Escherichia coli. The relationship between the bioactive properties of the fungus PBR.2.2.2 and those achieved for glandicoline B, as well the potential of this substance as bactericide is discussed.

An antimicrobial diketopiperazine alkaloid and co-metabolites from an endophytic strain of Gliocladium isolated from Strychnos cf. toxifera

From an endophytic strain of Gliocladium sp. isolated from the Amazonian plant Strychnos cf. toxifera, we obtained the diketopiperazine alkaloid cyclo-(glycyl-L-tyrosyl)-4,4-dimethylallyl ether (1), the steroids ergosterol (2), ergosterol peroxide (3), cerevisterol (4) and the citric acid (5). The AcOEt extract of the fermented broth by Gliocladium sp. showed potent activity against the cancer cell lines MDA-MB435 (human breast cancer cells), HCT-8 (human colorectal cancer cells) and SF-295 (human glioblastoma cancer cells). Compound 1 exhibited a strong antimicrobial activity against Micrococcus luteus at a concentration of 43.4 µM.

Synergy-directed fractionation of botanical medicines: a case study with goldenseal (Hydrastis canadensis).

It is often argued that the efficacy of herbal medicines is a result of the combined action of multiple constituents that work synergistically or additively. Determining the bioactive constituents in these mixtures poses a significant challenge. We have developed an approach to address this challenge, synergy-directed fractionation, which combines comprehensive mass spectrometry profiling with synergy assays and natural products isolation. The applicability of synergy-directed fractionation was demonstrated using the botanical medicine goldenseal (Hydrastis canadensis) as a case study. Three synergists from goldenseal were identified, sideroxylin, 8-desmethyl-sideroxylin, and 6-desmethyl-sideroxylin. These flavonoids synergistically enhance the antimicrobial activity of the alkaloid berberine (also a constituent of H. canadensis) against Staphylococcus aureus by inhibition of the NorA multidrug resistance pump. The flavonoids possess no inherent antimicrobial activity against S. aureus; therefore, they could have been missed using traditional bioactivity-directed fractionation. The flavonoid synergists are present at higher concentration in extracts from H. canadensis leaves, while the antimicrobial alkaloid berberine is present at higher levels in H. canadensis roots. Thus, it may be possible to produce an extract with optimal activity against S. aureus using a combination of goldenseal roots and leaves.

Synergy Directed Fractionation: A Case Study Identifying Efflux Pump Inhibitors from Goldenseal (Hydrastis canadensis)

It is often argued that herbal medicines are effective due to the combined effects of multiple chemical constituents that act synergistically. Identifying the constituents involved in these complex interactions continues to be a major hurdle in research with botanicals. Here we demonstrate an approach to address this problem, synergy directed fractionation. With this technique, crude extracts are screened prospectively for the presence of synergists. These extracts are then fractionated, and the fractions are again submitted to the synergy assay. Comprehensive profiling with liquid chromatography mass spectrometry (LC-MS) is used after each stage of fractionation as a central tool towards identifying possible active constituents. Bioactive compounds and synergists are ultimately isolated and their structures elucidated. This technique was applied to extracts prepared from the leaves of the widely used botanical goldenseal (Hydrastis canadensis L.). As a result, several flavonoids that have not previously been reported as constituents of H. canadensis were identified. The most abundant of these flavonoids, 8-demethly-sideroxylin (1), was shown to act as an inhibitor of the multidrug efflux pump NorA in Staphylococcus aureus. Importantly, this compound was only effective in combination with the antimicrobial alkaloid berberine (also produced by H. canadensis). Because the compound has no inherent antimicrobial activity, it would likely have been missed had the plant been subjected to traditional bioassay guided fractionation.

Antimicrobial Alkaloids from the Tubers of Stephania succifera

Chemical investigation of the EtOH extract from the tubers of Stephania succifera collected in Hainan Province of China resulted in the isolation of the two new alkaloids Cepharanone D (1) and N-formyl-asimilobine (2), and a known alkaloid, N-formyl-annonain (3). Their structures were elucidated by spectroscopic techniques (UV, IR, 1D and 2D NMR). The antimicrobial activities of the three compounds were also investigated.

Autoxidation of the Antimicrobial Alkaloid Cleistopholine in Solution.

The alkaloid cleistopholine (3) undergoes rapid autoxidation in ethyl acetate solution under air giving rise to the corresponding N-oxide on one pathway, and to oxidation of the methyl group to a hydroxymethyl, formyl and further on to a carboxy group on the other one. Autoxidation can be suppressed by protection from light and air. In methanolic solution the extent of oxidation is less, in hydrochloric acid solution it is neglectable. The quinone moiety seems to be involved in the disposition to undergo oxidation.

Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment.

Three key benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), were localized in cultured opium poppy (Papaver somniferum) cells by sucrose density gradient fractionation and immunogold labeling. CYP80B1 catalyzes the second to last step in the formation of (S)-reticuline, the last common intermediate in sanguinarine and morphine biosynthesis. BBE converts (S)-reticuline to (S)-scoulerine as the first committed step in sanguinarine biosynthesis, and COR catalyzes the penultimate step in the branch pathway leading to morphine. Sanguinarine is an antimicrobial alkaloid that accumulates in the vacuoles of cultured opium poppy cells in response to elicitor treatment, whereas the narcotic analgesic morphine, which is abundant in opium poppy plants, is not produced in cultured cells. CYP80B1 and BBE were rapidly induced to high levels in response to elicitor treatment. By contrast, COR levels were constitutive in the cell cultures, but remained low and were not induced by addition of the elicitor. Western blots performed on protein homogenates from elicitor-treated cells fractionated on a sucrose density gradient showed the cosedimentation of CYP80B1, BBE, and sanguinarine with calreticulin, and COR with glutathione S-transferase. Calreticulin and glutathione S-transferase are markers for the endoplasmic reticulum (ER) and the cytosol, respectively. In response to elicitor treatment, large dilated vesicles rapidly developed from the lamellar ER of control cells and fused with the central vacuole. Immunogold localization supported the association of CYP80B1 and BBE with ER vesicles, and COR with the cytosol in elicitor-treated cells. Our results show that benzylisoquinoline biosynthesis and transport to the vacuole are associated with the ER, which undergoes major ultrastructural modification in response to the elicitor treatment of cultured opium poppy cells.

A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy.

Opium poppy produces a diverse array of pharmaceutical alkaloids, including the narcotic analgesics morphine and codeine. The benzylisoquinoline alkaloids of opium poppy accumulate in the cytoplasm, or latex, of specialized laticifers that accompany vascular tissues throughout the plant. However, immunofluorescence labeling using affinity-purified antibodies showed that three key enzymes, (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), involved in the biosynthesis of morphine and the related antimicrobial alkaloid sanguinarine, are restricted to the parietal region of sieve elements adjacent or proximal to laticifers. The localization of laticifers was demonstrated using antibodies specific to the major latex protein (MLP), which is characteristic of the cell type. In situ hybridization showed that CYP80B1, BBE, and COR gene transcripts were found in the companion cell paired with each sieve element, whereas MLP transcripts were restricted to laticifers. The biosynthesis and accumulation of alkaloids in opium poppy involves cell types not implicated previously in plant secondary metabolism and dramatically extends the function of sieve elements beyond the transport of solutes and information macromolecules in plants.

Antimicrobial Activity of the Alkaloidal Constituents of the Root Bark of Eupomatia laurina

From the root bark of Eupomatia laurina were isolated four anti-microbial alkaloids; sampangine (I), eupomatine-1 (II), liriodenine and lanuginosine. The isolated alkaloids, the fraction from which I + II were isolated (EI), the dichloromethane fraction of the root bark (RD) and the ethyl acetate fraction of the stem bark (SE) were screened against 13 Gram + and 12 Gram - bacteria, a protozoan and nine fungi. All extracts were found to be active against all the bacteria and protozoan. Antifungal activity was demonstrated by EI, RD and SE fractions only. Compound II and fraction EI were found to be far superior to the standard used, both in broadness and level of activity. MIC was performed on EI and was found to be in most cases 0.5µg while in some case it was 0.1µg. Broad-spectrum antifungal activity was exhibited by fraction EI (10µg disc), RD (4 mg disc) and SE (4 mg disc). MIC was done on EI and was 10µg. The activity of EI, RD and SE was found to be far superior to the standard ketoconozole (20 mg disc) and griseofulvin (25 mg disc).

Cell type-specific protoberberine alkaloid accumulation in Thalictrum flavum

Summary Berberine is a common benzylisoquinoline alkaloid with potent antimicrobial properties, which suggest it functions to protect some plants from pathogen challenge. Berberine was identified as the major alkaloid in meadow rue (Thalictrum flavum ssp. glaucum), a medicinal member of the Ranunculaceae, and was detected in seeds and all organs of the plant. The high level of berberine in roots, rhizomes, and older petioles is mainly responsible for the intense yellow color of these organs. In rhizomes, protoberberine alkaloids were detected throughout the pith and, to a lesser extent, the cortex, but were absent from the vascular tissues. Similarly, protoberberine alkaloids were detected in the rib parenchyma of older petioles. In roots, alkaloid accumulation was detected only in the endodermis at the onset of secondary growth. Rather than being sloughed off, the endodermis was found to undergo extensive anticlinal division leading to an expanding cellular cylinder that ultimately displaced all external tissues. Endodermal-specific protoberberine alkaloid accumulation continued throughout root development, but was extended to include 3 to 4 layers of smaller pericycle cells in the oldest roots near the base of the stem. The cell type-specific accumulation of antimicrobial alkaloids and the unusual development of the endodermis and pericycle in T. flavum roots support the putative role of berberine in plant defense.

A short route to functionalized imidazo[4,5-c]carbazoles. Synthesis of the first example of the imidazo[4,5-c]β-carboline ring system

A new synthetic route to functionalized imidazo[4,5-c]carbazoles (4) via intramolecular electrocyclization of indolo-1,3,5-hexatriene system (3a) is described. Thermally induced ring-closure of 2-ethylcarboxylate-(4-amino-5-imidazolyl)-3-indole (3d) led to the previously unknown imidazo[4,5-c]β-carboline ring system (5). These heterocycles were efficiently converted into analogs of both the marine cytotoxic agents grossularines-1 and -2 (1) and the antimicrobial alkaloid eudistomin U (2).

Molecular cloning and functional expression of O-methyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis.

In cell suspension cultures of the meadow rue Thalictrum tuberosum, biosynthesis of the anti-microbial alkaloid berberine can be induced by addition of methyl jasmonate to the culture medium. The activities of the four methyltransferases involved in the formation of berberine from L-tyrosine are increased in response to elicitor addition. Partial clones generated by RT-PCR with methyltransferase-specific primers were used as hybridization probes to isolate four cDNAs encoding O-methyltransferases from a cDNA library prepared from poly(A)+ RNA isolated from methyl jasmonate-induced cell suspension cultures of T. tuberosum. RNA gel blot hybridization indicated that the transcripts for the methyltransferases accumulated in response to addition of methyl jasmonate to the cell culture medium. The cDNAs were functionally expressed in Spodoptera frugiperda Sf9 cells and were shown to have varying and broad substrate specificities. A difference of a single amino acid residue between two of the enzymes was sufficient to alter the substrate specificity. The four cDNAs were expressed either as four homodimers or as six heterodimers by co-infection with all possible combinations of the four recombinant baculoviruses. These 10 isoforms thus produced displayed distinct substrate specificities and in some cases co-infection with two different recombinant baculoviruses led to the O-methylation of new substrates. The substrates that were O-methylated varied in structural complexity from simple catechols to phenylpropanoids, tetrahydrobenzylisoquinoline, protoberberine and tetrahydrophenethylisoquinoline alkaloids, suggesting that some biosynthetic enzymes may be common to both phenylpropanoid and alkaloid anabolism.

Isolation and analysis of a gene bbe1 encoding the berberine bridge enzyme from the California poppy Eschscholzia californica.

A genomic clone, bbe1 was isolated that encodes the methyl jasmonate-inducible berberine bridge enzyme of antimicrobial benzophenanthridine alkaloid biosynthesis in the California poppy Eschscholzia californica. Genomic DNA gel blot analysis indicates that two genes are present in the E. californica genome that code for the berberine bridge enzyme reading frame. Each coding region is apparently preceeded by a unique promoter sequence. The bbe1 gene contains no introns and one transcriptional start site. A 41 nucleotide region between -496 and -455 of the 5'-flanking region appears to be essential for promoter activity in E. californica. The promoter displayed an unexpectedly high species specificity, being active in only E. californica and Thalictrum bulgaricum, out of 28 cell suspension cultures tested.

Molecular characterization of berberine bridge enzyme genes from opium poppy.

In Papaver somniferum (opium poppy) and related species, (S)-reticuline serves as a branch-point intermediate in the biosynthesis of numerous isoquinoline alkaloids. The berberine bridge enzyme (BBE) ([S]-reticuline:oxygen oxidoreductase [methylene bridge forming], EC 1.5.3.9) catalyzes the stereospecific conversion of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon of (S)-scoulerine and represents the first committed step in the pathway leading to the antimicrobial alkaloid sanguinarine. Three unique genomic clones (bbe1, bbe2, and bbe3) similar to a BBE cDNA from Eschscholtzia californica (California poppy) were isolated from opium poppy. Two clones (bbe2 and bbe3) contained frame-shift mutations of which bbe2 was identified as a putative, nonexpressed pseudogene by RNA blot hybridization using a gene-specific probe and by the lack of transient expression of a chimeric gene fusion between the bbe2 5' flanking region and a beta-glucuronidase reporter gene. Similarly, bbe1 was shown to be expressed in opium poppy plants and cultured cells. Genomic DNA blot-hybridization data were consistent with a limited number of bbe homologs. RNA blot hybridization showed that bbe genes are expressed in roots and stems of mature plants and in seedlings within 3 d after germination. Rapid and transient BBE mRNA accumulation also occurred after treatment with a fungal elicitor or with methyl jasmonate. However, sanguinarine was found only in roots, seedlings, and fungal elicitor-treated cell cultures.

Uncoupled defense gene expression and antimicrobial alkaloid accumulation in elicited opium poppy cell cultures.

Treatment of opium poppy (Papaver somniferum L.) cell cultures with autoclaved mycelial homogenates of Botrytis sp. resulted in the accumulation of sanguinarine. Elicitor treatment also caused a rapid and transient induction in the activity of tyrosine/dopa decarboxylase (TYDC, EC 4.1.1.25), which catalyzes the conversion of L-tyrosine and L-dopa to tyramine and dopamine, respectively, the first steps in sanguinarine biosynthesis. TYDC genes were differentially expressed in response to elicitor treatment. TYDC1-like mRNA levels were induced rapidly but declined to near baseline levels within 5 h. In contrast, TYDC2-like transcript levels increased more slowly but were sustained for an extended period. Induction of TYDC mRNAs preceded that of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) mRNAs. An elicitor preparation from Pythium aphanidermatum was less effective in the induction of TYDC mRNA levels and alkaloid accumulation; however, both elicitors equally induced accumulation of PAL transcripts. In contrast, treatment with methyl jasmonate resulted in an induction of TYDC but not PAL mRNAs. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and the protein kinase inhibitor staurosporine partially blocked the fungal elicitor-induced accumulation of sanguinarine. However, only staurosporine and okadaic acid, an inhibitor of protein phosphatases 1 and 2A, blocked the induction of TYDC1-like transcript levels, but they did not block the induction of TYDC2-like or PAL transcript levels. These data suggest that activation mechanisms for PAL, TYDC, and some later sanguinarine biosynthetic enzymes are uncoupled.

Biological Activities of Pyrrolidinoindoline Alkaloids fromCalycodendron milnei

Certain genera of the tribe Psychotrieae, specifically Calycodendron and Psychotria, found on Pacific Islands, synthesize a series of Nb-methyltryptamine-derived alkaloids made by linking together 2 to 8 pyrrolidinoindoline units. Nine alkaloids of this class have been isolated from the aerial parts and stem bark of Calycodendron milnei, a species endemic to the Vaté Islands (New Hebrides), and examined for potential application as anti-cancer and anti-infective agents. All members of the series exhibited readily detected cytotoxic activity against proliferating and non-proliferating Vero African green monkey kidney cells in culture, with the most potent activity being exhibited by vatamine and quadrigemine C. Only hodgkinsine A exhibited substantial antiviral activity against a DNA virus, herpes simplex type 1, and an RNA virus, vesicular stomatitis virus. All members of the series showed readily detected anti-bacterial, anti-fungal, and anti-candidal activities using both tube dilution and disc diffusion assay methods. The most potent anti-microbial alkaloids were hodgkinsine A and quadrigemine C, which exhibited minimum inhibitory concentration (MIC) values as low as 5 micrograms/ml.

Oceanapamine, a sesquiterpene alkaloid from the Philippine sponge Oceanapia sp.

The major metabolite of the Philippine sponge Oceanapia sp. is the antimicrobial alkaloid oceanapamine [1], which was isolated as a trifluoroacetate salt. The structure and the absolute configuration of oceanapamine, which consists of a monocyclic sesquiterpene attached to a histamine residue, were elucidated by interpretation of spectroscopic data.

Screening of Vernonia kotschyana for Antimicrobial Activity and Alkaloids

Vernonia kotschyana Schultz-Bip. (Compositae) is used in Kano, northern Nigeria, as a remedy for schomachache. Screening of the plant revealed antimicrobial activity against Gremmpositive and Gramnegative bacteria, including some bacterial species known to be etiological agents of gastrointestinal disorders. Moreover, the parent ethanol extracts of the plant were found to be bactericidal. Some fractions of the plant extracts tested positive for alkaloids which may contribute to the antibiosis of the plant.